Nucleotide Analogs

ABSTRACT

The invention provides for nucleotide analogs and methods of using the same, e.g., for sequencing nucleic acids.

RELATED APPLICATIONS

This application is a continuation-in-part (CIP) of U.S. applicationSer. No. 11/929,084 filed Oct. 30, 2007, which is a continuation of Ser.No. 11/803,339 filed May 5, 2007, which is a CIP of Ser. No. 11/603,945filed Nov. 22, 2006, which is a CIP of Ser. No. 11/295,406 filed Dec. 5,2007, which is a CIP of Ser. No. 11/286,626 filed Nov. 22, 2005; Ser.No. 11/803,339 filed May 14, 2007 is a CIP of Ser. No. 11/295,155 Dec.26, 2005, which is a CIP of Ser. No. 11/295,406 filed Dec. 5, 2005; Ser.No. 11/803,339 filed May 14, 2007 is a CIP of Ser. No. 11/496,262 filedJul. 31, 2006, which is a CIP of Ser. No. 11/295,155 filed Dec. 26,2005, which is a CIP of Ser. No. 11/295,406 filed Dec. 5, 2005; Ser. No.11/803,339 filed May 14, 2007 is a CIP of Ser. No. 11/496,274 filed Jul.31, 2006, which is a CIP of Ser. No. 11/496,262 filed Jul. 31, 2006;Ser. No. 11/603,945 filed Nov. 22, 2006 is a CIP of Ser. No. 11/496,275filed Jul. 31, 2006, which is a CIP of Ser. No. 11/496,274 filed Jul.31, 2006; this application is also a CIP of Ser. No. 11/137,928 filedMay 25, 2005 which claims priority to 60/574,389 filed May 25, 2004, theentire contents of each of the above applications are expresslyincorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The invention relates to nucleotide analogs and methods for sequencing anucleic acid using the nucleotide analogs.

BACKGROUND

Sequencing-by-synthesis involves the template-dependent addition ofnucleotides to a template/primer duplex. Traditionalsequencing-by-synthesis is performed using dye-labeled terminators andgel electrophoresis (so-called “Sanger sequencing”). See, e.g., Sanger,F. and Coulson, A. R., 1975, J. Mol. Biol. 94: 441-448; Sanger, F. etal., 1977, Nature. 265(5596): 687-695; and Sanger, F. et al., 1977,Proc. Natl. Acad. Sci. U.S.A. 75: 5463-5467. Recently, single moleculesequencing methods have been proposed that provide increased resolution,throughput, and speed at reduced cost. For example, asequencing-by-synthesis method that results in sequence determinationwithout consecutive base incorporation, has been proposed by Braslavsky,et al., Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003). These methods donot rely on the user of terminator nucleotides as in Sanger sequencing.Instead, template/primer duplex is anchored directly, or indirectly(e.g., via a polymerase enzyme) to a surface and labeled nucleotides areadded in a template-dependent manner.

A challenge that has arisen in single molecule sequencing involves theability to sequence through homopolymer regions (i.e., portions of thetemplate that contain consecutive identical nucleotides). Often thenumber of bases present in a homopolymer region is important from thepoint of view of genetic function. Many polymerase enzymes used insequencing-by-synthesis reactions are highly-processive and tend to addbases continuously in a homopolymer region. It is often difficult toresolve the number of nucleotides in a homopolymer due to the difficultyin distinguishing between the incorporation of one or two labelednucleotides and the incorporation of a greater number of nucleotides.

A need therefore exists for nucleotide analogs that promote accuratebase-over-base incorporation in sequencing-by-synthesis reactions.

SUMMARY OF THE INVENTION

The invention provides nucleotide analogs and methods of using them toallow sequencing-by-synthesis to occur such that, on average, a singlenucleotide is incorporated into the 3′ end of a primer portion of atemplate/primer duplex per sequencing cycle. The invention is based, inpart, on the discovery that nucleotide analogs having an attachedinhibitory region with one or more charged groups provide goodincorporation of a single nucleotide into the duplex without allowing asignificant, or any, amount of second, third, etc. base incorporation.

The invention generally provides nucleotide analogs and methods of usingnucleotide analogs in sequencing. More particularly, the inventionprovides compounds, methods and compositions useful in introduction of asingle base at a time in a template-dependent sequencing-by-synthesisreaction. The invention allows template-dependentsequencing-by-synthesis through all regions of a target nucleic acid,including homopolymer regions, and provides methods for thedetermination of the number of nucleotides present in a homopolymerregion.

The invention provides nucleotide analogs that comprise a nucleotide (ornucleotide analog), a detectable label, and an inhibitor group. Uponincorporation of the nucleotide, the inhibitor prevents subsequentnucleotide incorporation into the same duplex. However, upon removal ofthe detectable label and the inhibitor group, the nucleotide analog doesnot substantially hinder subsequent nucleotide (or nucleotide analog)incorporation.

In one aspect, A method for sequencing a nucleic acid. The methodincludes the steps of: exposing a nucleic acid duplex comprising atemplate portion and a primer portion to a nucleotide analog comprisingan inhibitor that is charged or capable of becoming charged, and apolymerase, under conditions that permit template-dependentincorporation of the analog into the primer; detecting incorporation ofthe analog; removing or neutralizing the inhibitor; and repeating theexposing, detecting, and removing steps at least once, thereby todetermine the sequence of the template.

In another aspect, the invention relates to a nucleotide analog thatincludes: a nucleoside triphosphate; an inhibitor comprising (a) one ormore multiply charged groups or groups capable of becoming multiplycharged, or (b) two or more (i.e., a plurality of) singly charged groupsor two or more groups capable of becoming singly charged; a detectablelabel; and a linker connecting the inhibitor and the label to thenucleoside triphosphate. It should be noted that in some embodiments,one or a single charged group may be sufficient to provide the desiredinhibitory effect.

In another aspect, the invention relates to nucleotide analogs of theformula:

NTP is a nucleoside or nucleotide triphosphate or an analog thereofcapable of template-dependent incorporation into the 3′ end of apolynucleotide strand hybridized to a template. Inhibitor comprises amoiety that is charged or capable of becoming charged and that inhibitssubsequent nucleotide incorporation once the first nucleotide isincorporated. Tether is a bond or a group linking the NTP to theInhibitor group. In a preferred embodiment, the inhibitor is anon-steric inhibitor.

In another aspect, the invention relates to nucleotide analogs ofFormula II:

NTP is a nucleoside or nucleotide triphosphate or an analog of eithercapable of template-dependent incorporation into the 3′ end of apolynucleotide strand hybridized to a template presenting the complementof the NTP. L is a detectable label that facilitates the identificationof the nucleotide analog. Inhibitor comprises (a) one or more multiplycharged groups or groups capable of becoming multiply charged, or (b)two or more singly charged groups or two or more groups capable ofbecoming singly charged. R₁ and R₂ are independently a bond or a group,wherein at least one of R₁ and R₂ comprises a cleavable bond, which uponcleavage results in de-association of NTP from both Label and Inhibitor.R₃ is a bond or group linking R₂ to the Inhibitor. R₄ is a bond or grouplinking R₂ to a Label.

In another aspect, the invention relates to a method for sequencing anucleic acid. The method includes: (a) anchoring a nucleic acid duplex,or portion thereof, to a surface, the duplex comprising a templateportion and a primer portion hybridized thereto; (b) exposing the duplexto nucleotide analog of Formula I or II (as defined herein) in thepresence of a polymerase capable of catalyzing the addition of thenucleotide analog to the primer portion in a template-dependent manner;(c) removing unincorporated nucleotide analog and polymerase; (d)detecting incorporation of the nucleotide analog into the primerportion; and repeating the exposing, removing, and detecting steps atleast once.

In another aspect, the invention provides methods and nucleotide analogsfor selectively inhibiting the catalytic function of a polymeraseenzyme. As such, nucleotide analogs comprise an inhibitory portion, suchthat the nucleotide analog is capable of being incorporated into anucleic acid duplex but then inhibits subsequent nucleotideincorporation until the inhibitory portion is removed.

The inhibitory portion of an analog of the invention preferably is acharged group. The charged group can take any appropriate form as longas it carries a charge. Preferably, the charge group is selected from aphosphate, a carboxylic acid (or carboxylate), a sulfate, caproic acid(or a caproic acid derivative), a charged amino acid, —SO₃, —SO₂, and—NR_(w)R_(v), where R_(w) and R_(v) independently is H, an alkyl or arylgroup. The charged group can convey a negative or positive charge, butnegative charged groups are preferred. In another preferred embodiment,the charge group contains multiple charged portions. For example, thecharge group can be a dipeptide, a di-phosphate, disulfate, or othermultiples of charged moieties. For example, amino acid inhibitors arepreferably selected from aspartic acid, glutamic acid, arginine, lysine,and histidine.

The invention provides charged inhibitors of subsequent baseincorporation in a sequencing-by-synthesis reaction. By subsequent baseincorporation it is intended that a first nucleotide (or analog) isincorporated in a template-dependent manner, but second, third, etc.base incorporation is inhibited by the inhibitor group. In a preferredembodiment, inhibition occurs by positioning a charged group inproximity to the active site of a polymerase enzyme, thus disabling theability of the polymerase to make subsequent incorporations. Withoutbeing limited to theory, analogs of the invention, interfere withmagnesium present in the active site of the polymerase, resulting in areduced ability of the active site to catalyze subsequent nucleotideincorporation.

In a preferred embodiment, an analog of the invention comprises anucleoside triphosphate, an inhibitor comprising a plurality of chargedgroups, a detectable label, and a linker connecting the charged groupsand the label to the nucleoside triphosphate. Preferred inhibitorscomprise a plurality of charged groups and may be selected from anycharged group capable of conferring a charge in a local area.Preferably, the inhibitor does not sterically inhibit a polymerase. Alsoin a preferred embodiment, the linker is cleavable. Multiple cleavablegroups, such as enzymatically-cleavable group, such as disulfide bondsand the like.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions that facilitate theaddition of a single nucleotide to a template/primer duplex per reactioncycle (i.e., the addition of nucleotides and polymerase enzyme underconditions that result in template-dependent nucleotide incorporationinto the primer). Analogs of the invention comprise a charged inhibitorygroup that, upon incorporation of a nucleotide in a template-dependentmanner, prevents subsequent nucleotide incorporation until theinhibitory group is removed. Thus, an analog of the invention comprisesa nucleotide triphosphate, a linker (or tether), a detectable label, anda charged inhibitory group, wherein the label and the inhibitory groupare removable.

In one aspect, the invention generally provides nucleotide analogs ofthe following Formula I:

whereinNTP is a nucleoside triphosphate or an analog thereof capable ofincorporating onto the 3′ end of a polynucleotide strand hybridized to atemplate presenting the complement of the NTP;Inhibitor comprises a group that is charged or capable of becomingcharged, e.g., under reaction conditions, and that inhibits a subsequentincorporation of a nucleotide (or analog thereof), andTether is a bond or a group linking the NTP to the Inhibitor moiety. Agroup is considered capable of becoming charged if the group is capableof becoming electrically non-neutral, e.g., under reaction or bufferconditions. Examples of such groups include —COOH and —NR_(w)R_(v),where R_(w), and R_(v), independently is H, an alkyl or aryl group.

In one embodiment, the inhibitor group can cause inhibition ofsubsequent nucleotide incorporation without steric hinderance. In otherwords, the inhibition is caused by chemical or charge interaction withthe enzyme and not be a physical blocking of the enzyme. In anotherembodiment, the charged inhibitor also provides steric inhibition ofenzyme activity. However, in either case, the inhibitor group ischarged.

Natural NTPs include nucleoside triphosphates, adenosine triphosphate(ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP),thymidine triphosphate (TTP) and uridine triphosphate (UTP); andnucleotide triphosphates, deoxyadenosine triphosphate (dATP),deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP),deoxythimidine triphosphate (dTTP) and deoxyuridine triphosphate (dUTP).NTPs useful in this invention include non-nature nucleosides andnucleotides, and analogs and derivatives thereof.

In some embodiments, the inhibitor may include a moiety that isnegatively charged or capable of becoming a negatively charged. In otherembodiments, the inhibitor group is positively charged or capable ofbecoming positively charged.

In some other embodiments, the inhibitor is an amino acid or an aminoacid analog. The Inhibitor may be a peptide of 2 to 20 units of aminoacids or analogs, a peptide of 2 to 10 units of amino acids or analogs,a peptide of 3 to 7 units of amino acids or analogs, a peptide of 3 to 5units of amino acids or analogs. In some embodiments, the Inhibitorincludes a group selected from the group consisting of Glu, Asp, Arg,His, and Lys, and a combination thereof (e.g., Arg, Arg-Arg, Asp,Asp-Asp, Asp, Glu, Glu-Glu, Asp-Glu-Asp, Asp-Asp-Glu or AspAspAspAsp).Peptides or groups may be combinations of the same or different aminoacids or analogs.

In one embodiment, the invention relates to an oligonucleotide with atleast one nucleotide analog of the invention incorporated therein.

In some embodiments, the Tether comprises

wherein L is detectable label that facilitates the identification of thenucleotide analog after incorporation onto a template;R₁ and R₂ are independently a bond or a group, wherein at least one ofR₁ and R₂ comprises a cleavable bond, which upon cleavage results inde-association of NTP from both L and Inhibitor;R₃ is a bond or group linking R₂ to the Inhibitor moiety; andR₄ is a bond or group linking R₂ to a L.

In another aspect, the present invention is directed to nucleotideanalogs of Formula II:

whereinNTP is a nucleoside triphosphate or an analog thereof capable ofincorporating onto the 3′ end of a polynucleotide strand hybridized to atemplate presenting the complement of the NTP;L is a detectable label to facilitate the identification of thenucleotide analog after incorporation onto the template;Inhibitor is a moiety that substantially inhibits a subsequentincorporation of a nucleotide (or analog thereof). In some embodiments,the Inhibitor moiety includes a nucleotide or nucleoside or analogsthereof, in other embodiments, the inhibitor is not a nucleotide oranalog thereof;R₁ and R₂ are independently a bond or a group, wherein at least one ofR₁ and R₂ comprises a cleavable bond, which upon cleavage results inde-association of NTP from both Label and Inhibitor;R₃ is a bond or group linking R₂ to the Inhibitor moiety; andR₄ is a bond or group linking R₂ to L.

In some embodiments, NTP is a compound having the following formula:

wherein B¹ is selected from the group consisting of purine or pyrimidinebases, as well as derivatives of purine and pyrimidine bases; R′ isindependently selected from the group consisting of —OH, —O—P(O)(OH)₂,—O—C(O)—Rx, —NHR^(y), and an —O-blocking agent, where R^(x) and R^(y)are alkyl groups; R″ is independently selected from the group consistingof H and —OH.

Non-limiting examples of representative purine and pyrimidine basesinclude adenine, cytosine, guanine, thymine, uracil, or hypoxanthine.Non-limiting examples of derivatives of purine and pyrimidine basesinclude naturally-occurring and synthetic derivatives of a base,including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine,7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine.

Base B¹ of the invention permits a nucleotide to be incorporated into apolynucleotide chain by a polymerase and forms base pairs with a base onan antiparallel nucleic acid strand. The term base pair encompasses notonly the standard AT, AU or GC base pairs, but also base pairs formedbetween nucleotides and/or nucleotide analogs comprising non-standard ormodified bases, wherein the arrangement of hydrogen bond donors andhydrogen bond acceptors permits hydrogen bonding between a nonstandardbase and a standard base or between two complementary non-standard basestructures. One example of such non-standard base pairing is the basepairing between the nucleotide analog inosine and adenine, cytosine oruracil, where two hydrogen bonds are formed.

The Inhibitor may include a charged moiety (e.g., a negatively chargedmoiety, a positively charged moiety, or both) or a moiety that iscapable of becoming charged. The Inhibitor can include two or morecharged groups. The Inhibitor may have a charged group selected from thegroup consisting of —COOH, —PO₄, —SO₄, —SO₃, —SO₂, —NR_(w)R_(v), whereR_(w) and R_(v) independently is H, an alkyl or aryl group. In otherembodiments, the Inhibitor moiety does not comprise a —PO₄ group. Insome other embodiments, the Inhibitor moiety does not comprise an arylgroup. In certain other embodiments, the Inhibitor does not include anucleotide or nucleoside or analogs thereof.

Inhibitor may be a compound having the following formula:

wherein R₈ and R₉ independently is a H or an alkyl group; each of x andy is an integer from 0 to about 5. In some embodiments, R₈ and R₉ are Hatoms and x=1 and y=2.

R₃ of a nucleotide analog of Formula II may include a group having theformula of

wherein R₅ is a H or an alkyl group; p is an integer from 0 to about 10.In some embodiments, p is 5 or 6.

In some embodiments, R₃ of a nucleotide analog of Formula II may includea group having the formula of

wherein k is an integer from about 1 to about 5. In some embodiments, kis an integer from about 2 to about 4. In some embodiments, k is 3.

In some embodiments, R₃ of a nucleotide analog of Formula II may includea group having the formula of

wherein R¹, R² are independently H or alkyl groups, and may togetherform one or more 3, 4, 5, or 6-member rings, and j is an integer fromabout 1 to about 5. In some embodiments, R₃ of include a group havingthe formula of

In some embodiments, R₁ of a nucleotide analog of Formula II may includea group having the formula of

wherein R¹, R², R³, and R⁴ are independently H or alkyl groups, and twoor more of which may together form one or more 3, 4, 5, or 6-memberrings, and j is an integer from about 1 to about 3. In some embodiments,R₁ of include a group having the formula of

R¹ of a nucleotide analog of Formula II may include a C—C triple bond, aS—S bond, or both a C—C triple bond and a S—S bond.

In some embodiments, R₁ in the nucleotide analog of Formula II includesa group having the formula of

wherein R₆ is a H or an alkyl group; q and r independently is an integerfrom about 1 to about 10.

In some embodiments, q is 1 or 2 and r is 1, 2 or 3.

In some embodiments of the invention, the location of the charged moietywithin the inhibitor group and/or the distance of the charged group tothe NTP plays an important role in the effectiveness of inhibiting asubsequent nucleotide incorporation. In some embodiments, the chargedmoiety of the inhibitor is from about 5 to about 60 bonds away from theNTP. In some other embodiments, the charged moiety of the inhibitor isfrom about 10 to about 40 bonds away from the NTP. In some otherembodiments, the charged moiety of the inhibitor is from about 10 toabout 35 bonds away from the NTP. In some other embodiments, the chargedmoiety of the inhibitor is from about 10 to about 30 bonds away from theNTP. In some other embodiments, the charged moiety of the inhibitor isfrom about 10 to about 20 bonds away from the NTP.

For example, the above compound (about 17× fold inhibition) exhibits aninhibiting effect that is much less than the following compound (about70× fold inhibition).

The label (or “L”) may be any moiety that can be attached to orassociated with, e.g., directly or via a linker or spacer, anoligonucleotide and that functions to provide a detectable signal,and/or to interact with a second label to modify the detectable signalprovided by the first or second label, e.g. fluorescence resonanceenergy transfer (FRET). In one embodiment, the label is anoptically-detectable moiety (e.g., a fluorophore). Non-limiting examplesof types of optically-detectable labels include a fluorescent,chemiluminescence, or electrochemically luminescent label. Examples offluorescent labels include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives thereof such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 15 1);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylaminolnaphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalocyanine; naphthalocyanine; any of the fluorescentlabels available from Atto-Tec, such as Atto 390, Atto 425, Atto 465,Atto 488, Atto 495, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590,Atto 594, Atto 610, Atto 611X, Atto 620, Atto 633, Atto 635, Atto 637,Atto 647, Atto 647N, Atto 655, Atto 680, Atto 700, Atto 725, Atto 740,etc.; any of the fluorescent labels available from Dyomics such asDY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, Dy-647, Dy-648,DY-649, Dy-650, Dy-651, DY-652, etc.; any of the fluorescent labelsavailable from Pierce such as DyLight 405, DyLight 488, DyLight 549,DyLight 633, DyLight 649, DyLight 680, DyLight 800, etc.; any of thefluorescent labels available from AnaSpec such as HiLyte Fluor™ 488dyes, HiLyte Fluor™ 555 dyes, HiLyte Fluor™ 647 dyes, HiLyte Fluor™ 680dyes, HiLyte Fluor™ 750 dyes, HiLytePlus™ 555 dyes, HiLytePlus™ 647dyes, HiLytePius™ 750 dyes, etc.; any of the fluorescent labelsavailable from Denovo Biolables such as Oyster 500, Oyster 550 P, Oyster550 D, Oyster 556, Oyster 645, Oyster 650 P, Oyster 650 D, Oyster 656,etc.; IRDye® 680, IRDye® 700, IRDye® 700DX, IRDye® 800, IRDye® 800 RS,IRDye® 800 CW, etc.; any of the fluorescent labels available from SETABiomedicals such as Seta K1-204, Seta K5-3212, Seta K8-1342, SetaK8-1352, Seta K8-1357, Seta K8-1407, Seta K8-1642, Seta K8-1644, SetaK8-1663, Seta K8-1664, Seta K8-1669, Seta K8-3002, Seta K4-1082, SetaK8-1669, Seta K7-545, Seta K7-547, Seta K7-549, Seta K8-1252, SetaK8-1261, Seta K8-1262, Seta K8-1320, Seta K8-1344, Seta K8-1367, SetaK8-1377, Seta K8-1382, Seta K8-1446, Seta K8-1667, Seta K8-1752, SetaK8-1762, Seta K8-1767, Seta K8-1777, Seta K8-1782, etc.; Q Dots; anddyes having the following structures:

wherein each Rx is independently selected from the group consisting ofH, alkyl, and substituted alkyl.

The above exemplary label moieties include any derivatives containingthe chromophore of any of the labeling moieties exemplified or describedherein, attached to the nucleotide analog by means of any suitablechemical linking group. For example, the chromophore can be attached tothe nucleotide analog via an alkyl chain bonded to the nucleotide analogby a functional group such as an amide, ester, ether, amine, thiol,disulfide, urea, urethane, carbonate, etc. In one embodiment, the labelis a fluorescent label such as cyanine-3 and cyanine-5.

Labels other than fluorescent labels are contemplated as part of theinvention, including other optically-detectable labels. Any appropriatedetectable label can be used according to the invention, and numerousother labels are known to those skilled in the art.

The invention also relates to methods for nucleic acid sequencedetermination using the nucleotide analogs described herein. Thenucleotide analogs of the invention are particularly suitable for use insingle molecule sequencing techniques. Such techniques are described forexample in U.S. patent application Ser. Nos. 10/831,214 filed April2004; 10/852,028 filed May 24, 2004; 10/866,388 filed Jun. 10, 2005;10/099,459 filed Mar. 12, 2002; and U.S. Published Application2003/013880 published Jul. 24, 2003, each of which is hereinincorporated in its entirety for all purposes. In general, methods fornucleic acid sequence determination include exposing a target nucleicacid (also referred to herein as template nucleic acid or template) to aprimer that is complementary to at least a portion of the target nucleicacid, under conditions suitable for hybridizing the primer to the targetnucleic acid, forming a template/primer duplex.

The invention also relates to methods for nucleic acid sequencedetermination using the nucleotide analogs described herein. Thenucleotide analogs of the invention are particularly suitable for use insingle molecule sequencing techniques. Such techniques are described forexample in U.S. patent application Ser. Nos. 10/831,214 filed April2004; 10/852,028 filed May 24, 2004; 10/866,388 filed Jun. 10, 2005;10/099,459 filed Mar. 12, 2002; and U.S. Published Application2003/013880 published Jul. 24, 2003, each of which is hereinincorporated in its entirety for all purposes. In general, methods fornucleic acid sequence determination include exposing a target nucleicacid (also referred to herein as template nucleic acid or template) to aprimer that is complementary to at least a portion of the target nucleicacid, under conditions suitable for hybridizing the primer to the targetnucleic acid, forming a template/primer duplex.

In another aspect, the invention relates to a method for sequencing anucleic acid. The method includes: (a) anchoring a nucleic acid duplexto a surface, the duplex comprising a template portion and a primerportion hybridized thereto; (b) exposing the duplex to nucleotide analogof Formula I or Formula II in the presence of a polymerase capable ofcatalyzing the addition of the nucleotide analog to the primer portionin a template-dependent manner; (c) removing unincorporated nucleotideanalog and polymerase; (d) detecting incorporation of the nucleotideanalog into the primer portion; and (e) repeating said exposing,removing, and detecting steps at least once. The method may furtherinclude cleaving L from the nucleotide analog after the detecting step.

In another aspect, the invention relates to a method for inhibiting thecatalytic function of a polymerase enzyme in a sequencing-by-synthesisreaction comprising introducing a nucleotide attached to an inhibitorygroup. In one aspect, the invention comprises attaching one or bothmembers of a template/primer duplex to a surface, introducing apolymerase and a nucleotide analog comprising a charged inhibitor underconditions sufficient for template-dependent incorporation of thenucleotide and inhibition of subsequent incorporation. Such methodsfurther comprise removing or neutralizing the inhibitor in order tofacilitate further nucleotide incorporation. Finally, nucleotides of theinvention can be detectably labeled to monitor incorporation.

Target nucleic acids include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Target nucleic acid molecules can be obtainedfrom any cellular material obtained from an animal, plant, bacterium,virus, fungus, or any other cellular organism, or may be synthetic DNA.Target nucleic acids may be obtained directly from an organism or from abiological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue.Any tissue or body fluid specimen may be used as a source for nucleicacid for use in the invention. Nucleic acid molecules may also beisolated from cultured cells, such as a primary cell culture or a cellline. The cells from which target nucleic acids are obtained can beinfected with a virus or other intracellular pathogen. Nucleic acidmolecules may also include those of animal (including human), wild typeor engineered prokaryotic or eukaryotic cells, viruses or completely orpartially synthetic RNAs or DNAs. A sample can also be total RNAextracted from a biological specimen, a cDNA library, or genomic DNA.

Nucleic acid typically is fragmented to produce suitable fragments foranalysis. In one embodiment, nucleic acid from a biological sample isfragmented by sonication. Test samples can be obtained as described inU.S. Patent Application 2002/0190663 A1, published Oct. 9, 2003, hereinincorporated by reference in its entirety for all purposes. Generally,nucleic acid can be extracted from a biological sample by a variety oftechniques such as those described by Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281(1982). Generally, target nucleic acid molecules can be from about 5bases to about 20 kb, about 30 kb, or even about 40 kb or more. Nucleicacid molecules may be single-stranded, double-stranded, ordouble-stranded with single-stranded regions (for example, stem- andloop-structures)

Single molecule sequencing includes a template nucleic acidmolecule/primer duplex that is immobilized on a surface such that theduplex and/or the nucleotides (or nucleotide analogs) added to theimmobilized primer are individually optically resolvable. The primer,template and/or nucleotide analogs are detectably labeled such that theposition of an individual duplex molecule is individually opticallyresolvable. Either the primer or the template is immobilized to a solidsupport. The primer and template can be hybridized to each other andoptionally covalently cross-linked prior to or after attachment ofeither the template or the primer to the solid support.

In general, methods for facilitating the incorporation of a nucleotideanalog as an extension of a primer include exposing a target nucleicacid/primer duplex to one or more nucleotide analogs disclosed hereinand a polymerase under conditions suitable to extend the primer in atemplate dependent manner. Generally, the primer is sufficientlycomplementary to at least a portion of the target nucleic acid tohybridize to the target nucleic acid and allow template-dependentnucleotide polymerization. The primer extension process can be repeatedto identify additional nucleotide analogs in the template. The sequenceof the template is determined by compiling the detected nucleotides,thereby determining the complementary sequence of the target nucleicacid molecule.

Any polymerase and/or polymerizing enzyme may be employed. A preferredpolymerase is Klenow with reduced exonuclease activity. Nucleic acidpolymerases generally useful in the invention include DNA polymerases,RNA polymerases, reverse transcriptases, and mutant or altered forms ofany of the foregoing. DNA polymerases and their properties are describedin detail in, among other places, DNA Replication 2nd edition, Kombergand Baker, W. H. Freeman, New York, N.Y. (1991). Known conventional DNApolymerases useful in the invention include, but are not limited to,Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene,108: 1, Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaelset al., 1996, Biotechniques, 20: 186-8, Boehringer Mannheim), Thermusthermophilus (Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh andMcGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis(Tli) DNA polymerase (also referred to as Vent™ DNA polymerase, Carielloet al., 1991, Polynucleotides Res, 19: 4 193, New England Biolabs),9″Nm™ DNA polymerase (New England Biolabs), Stoffel fragment,Thermosequenase® (Amersham Pharmacia Biotech UK), Therminator™ (NewEngland Biolabs), Thermotoga maritima (Tma) DNA polymerase (Diaz andSabino, 1998 Braz J. Med. Res, 3 1:1239), Thermus aquaticus (Taq) DNApolymerase (Chien et al., 1976, J. Bacteoriol, 127: 1550), DNApolymerase, Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al.,1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (fromthermococcus sp. JDF-3, Patent application WO 0132887), Pyrococcus GB-D(PGB-D) DNA polymerase (also referred as Deep VentTMD NA polymerase,Juncosa-Ginesta et al., 1994, Biotechniques, 16:820, New EnglandBiolabs), UITma DNA polymerase (from thermophile Thermotoga maritima;Diaz and Sabino, 1998 Braz J. Med. Res, 3 1: 1239; PE AppliedBiosystems), Tgo DNA polymerase (from thermococcus gorgonarius, RocheMolecular Biochemicals), E. coli DNA polymerase I (Lecomte andDoubleday, 1983, Polynucleotides Res. 11:7505), T7 DNA polymerase(Nordstrom et al., 198 1, J. Biol. Chem. 256:3 1 12), and archaealDP1I/DP2 DNA polymerase II (Cann et al., 1998, Proc Natl Acad. Sci. USA95: 14250-5).

Other DNA polymerases include, but are not limited to, ThermoSequenase®,9° Nm™, Therminator™, Taq, Tne, Tma, Pfu, Tfl, Tth, Tli, Stoffelfragment, Vent™ and Deep Vent™ DNA polymerase, KOD DNA polymerase, Tgo,JDF-3, and mutants, variants and derivatives thereof. Reversetranscriptases useful in the invention include, but are not limited to,reverse transcriptases from HIV, HTLV-1, HTLV-11, FeLV, FIV, SIV, AMV,MMTV, MoMuLV and other retroviruses (see Levin, Cell 88:5-8 (1997);Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et al., CRC Crit. RevBiochem. 3:289-347 (1975)).

Unincorporated nucleotide analog molecules may be removed prior to orafter detecting. Unincorporated nucleotide analog molecules may beremoved by washing.

A template/primer duplex is treated to remove the label and/or to cleavethe molecular chain attaching the label to the nucleotide. One mayrepeat the steps of exposing template/primer duplex to one or morenucleotide analogs and polymerase, detecting incorporated nucleotides,and then treating to (1) remove the label, (2) remove the label and atleast a portion of the molecular chain associating the label to thenucleotide or (3) cleave the molecular chain thereby identifyingadditional bases in the template nucleic acid, The identified bases canbe compiled to determine the sequence of the target nucleic acid. Insome embodiments, at least some portions of the remaining molecularchain and/or label are not removed, for example, in the last round ofprimer extension.

In some embodiments, a nucleotide analog, after removal of the label andportions of the molecular chain connecting the label to the nucleotidecan be represented by:

wherein B¹, R′, R″, are as described herein, and z is an integer fromabout 1 to about 12. R⁷ is a phosphodiester linkage connecting thenucleotide analog to a sugar of an adjacent nucleotide in the nucleicacid, or a phosphoryl group. In some embodiments, z is an integer fromabout 1 to about 5. In some other embodiments, z is an integer fromabout 1 to about 3.

The invention also provides for a method of removing a label from alabeled base, comprising (a) exposing a base of Formula I or Formula II:

as described herein, to a reducing agent for a time sufficient toproduce an unlabelled base of Formula III:

where B¹ is a part of the NTP of a nucleotide analog in Formula I orFormula II, and n is an integer from about 1 to about 12. In someembodiments, the reducing agent is tris (2-carboxyl ethyl) phosphine. Inother embodiments, the base is linked to a sugar selected from the groupconsisting of ribose, deoxyribose, and analogs thereof, where the baseand sugar together may be present in a nucleotide in a nucleic acid.

One embodiment of a method for sequencing a nucleic acid templateincludes exposing a nucleic acid template to a primer capable ofhybridizing to the template, a polymerase capable of catalyzingnucleotide addition to the primer, and a labeled nucleotide analogdisclosed herein under conditions to permit the polymerase to add thenucleotide analog to the primer. A method for sequencing may furtherinclude identifying or detecting the incorporated labeled nucleotide. Acleavable bond may then be cleaved, removing at least the label from thenucleotide analog. The exposing, detecting, and removing steps arerepeated at least once. In certain embodiments, the exposing, detecting,and removing steps are repeated at least three, five, ten or even moretimes. The sequence of the template can be determined based upon theorder of incorporation of the labeled nucleotides.

In another embodiment, a method for sequencing a nucleic acid templateincludes exposing a nucleic acid template to a primer capable ofhybridizing to the template and a polymerase capable of catalyzingnucleotide addition to the primer. The polymerase is, for example,Klenow with reduced exonuclease activity. The polymerase adds a labelednucleotide analog disclosed herein. The method may include identifyingthe incorporated labeled nucleotide. Once the labeled nucleotide isidentified, the label and at least a portion of a molecular chainconnecting the label to the nucleotide analog are removed and theremaining portion of the molecular chain includes a free hydroxyl group.The exposing, incorporating, identifying, and removing steps arerepeated at least once, preferably multiple times depending on theapplication. The sequence of the template is determined based upon theorder of incorporation of the labeled nucleotides.

Removal of a label from a labeled nucleotide analog and/or cleavage ofthe molecular chain linking a nucleotide analog to a label may includecontacting or exposing the labeled nucleotide with a reducing agent.Such reducing agents include, for example, dithiothreitol (DTT),tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxy-propyl) phosphine,tris(2-chloropropyl) phosphate (TCPP), 2-mercaptoethanol,2-mercaptoethylamine, cystein and ethylmaleimide. Such contacting orexposing the reducing agent to a labeled nucleotide analog may occur ata range of pH values, for example at a pH of about 5 to about 10, orabout 7 to about 9.

The above-described methods for sequencing a nucleic acid template canfurther include a step of capping a molecular chain, for example, afterthe label has been removed. After addition of the nucleotide analog tothe primer, any optional 3′ phosphate moiety can be removedenzymatically. In one embodiment, an optional phosphate can be removedusing alkaline phosphatase or T₄ polynucleotide kinase. Suitable enzymesfor removing optional phosphate include, any phosphatase, for example,alkaline phosphatase such as shrimp alkaline phosphatase, bacterialalkaline phosphatase, or calf intestinal alkaline phosphatase.

Any suitable detection method may be used to identify an incorporatednucleotide analog. Thus, exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. Single-molecule fluorescence can be carried out usinga conventional microscope equipped with total internal reflection (TIR)objective. The detectable moiety associated with the extended primerscan be detected on a substrate by scanning all or portions of eachsubstrate simultaneously or serially, depending on the scanning methodused. For fluorescence labeling, selected regions on a substrate may beserially scanned one-by-one or row-by-row using a fluorescencemicroscope apparatus, such as described in Fodor (U.S. Pat. No.5,445,934) and Mathies et al. (U.S. Pat. No. 5,091,652). Devices capableof sensing fluorescence from a single molecule include scanningtunneling microscope (STM) and the atomic force microscope (AFM).Hybridization patterns may also be scanned using a CCD camera (e.g.,Model TE/CCD512SF, Princeton Instruments, Trenton, N.J.) with suitableoptics (Ploem, CCD (Chase-Completed-Device) in Fluorescent andLuminescent Probes for Biological Activity Mason, T. G. Ed., AcademicPress, Landon, pp. 1-11 (1993), such as described in Yershov et al.,Proc. Natl. Aca. Sci. 93:4913 (1996), or may be imaged by TV monitoring.For radioactive signals, a phosphorimager device can be used (Johnstonet al., Electrophoresis, 13566, 1990; Drmanac et al., Electrophoresis,13:566, 1992; 1993). Other commercial suppliers of imaging instrumentsinclude General Scanning Inc., (Watertown, Mass. on the World Wide Webat genscan.com), Genix Technologies (Waterloo, Ontario, Canada; on theWorld Wide Web at confocal.com), and Applied Precision Inc. Suchdetection methods are particularly useful to achieve simultaneousscanning of multiple attached target nucleic acids.

The present invention provides for detection of molecules ranging from asingle nucleotide to a single target nucleic acid molecule. A number ofmethods are available for this purpose. Methods for visualizing singlemolecules within nucleic acids labeled with an intercalating dyeinclude, for example, fluorescence microscopy. For example, thefluorescent spectrum and lifetime of a single molecule excited-state canbe measured. Standard detectors such as a photomultiplier tube oravalanche photodiode can be used. Full field imaging with a two-stageimage intensified CCD camera also can be used. Additionally, low noisecooled CCD can also be used to detect single fluorescent molecules.

The detection system for the signal may depend upon the labeling moietyused. For optical signals, a combination of an optical fiber or chargecoupled device (CCD) can be used in the detection step. In thosecircumstances where the substrate is itself transparent to the radiationused, it is possible to have an incident light beam pass through thesubstrate with the detector located opposite the substrate from thetarget nucleic acid. For electromagnetic labeling moieties, variousforms of spectroscopy systems can be used. Various physical orientationsfor the detection system are available and discussion of designparameters is provided in the art.

A number of approaches can be used to detect incorporation offluorescently labeled nucleotides into a single nucleic acid molecule.Optical setups include near-field scanning microscopy, far-fieldconfocal microscopy, wide-field epi-illumination, but are not limitedto, light scattering, dark field microscopy, photoconversion, singleand/or multiphoton excitation, spectral wavelength discrimination,fluorophore identification, evanescent wave illumination, and totalinternal reflection fluorescence (TIRF) microscopy. In general, certainmethods involve detection of laser-activated fluorescence using amicroscope equipped with a camera. Suitable photon detection systemsinclude, but are not limited to, photodiodes and intensified CCDcameras. For example, an intensified charge couple device (ICCD) cameracan be used. The use of an ICCD camera to image individual fluorescentdye molecules in a fluid near a surface provides numerous advantages.For example, with an ICCD optical setup, it is possible to acquire asequence of images (movies) of fluorophores.

Some embodiments of the present invention use TIRF microscopy fortwo-dimensional imaging. TIRF microscopy uses totally internallyreflected excitation light and is well known in the art. See, e.g., theWorld Wide Web at nikoninstrurnents.jp/eng/page/products/tirf.aspx. Incertain embodiments, detection is carried out using evanescent waveillumination and total internal reflection fluorescence microscopy. Anevanescent light field can be set up at the surface, for example, toimage fluorescently-labeled nucleic acid molecules. When a laser beam istotally reflected at the interface between a liquid and a solidsubstrate (e.g., a glass), the excitation light beam penetrates only ashort distance into the liquid. The optical field does not end abruptlyat the reflective interface, but its intensity falls off exponentiallywith distance. This surface electromagnetic field, called the“evanescent wave”, can selectively excite fluorescent molecules in theliquid near the interface. The thin evanescent optical field at theinterface provides low background and facilitates the detection ofsingle molecules with high signal-to-noise ratio at visible wavelengths.

The evanescent field also can image fluorescently-labeled nucleotidesupon their incorporation into the attached target nucleic acid targetmolecule/primer complex in the presence of a polymerase. Total internalreflectance fluorescence microscopy is then used to visualize theattached target nucleic acid target molecule/primer complex and/or theincorporated nucleotides with single molecule resolution.

Fluorescence resonance energy transfer (FRET) can be used as a detectionscheme. FRET in the context of sequencing is described generally inBraslavasky, et al., Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003),incorporated by reference herein. In an embodiment, a donor fluorophoreis attached to the primer, polymerase, or template. Nucleotides addedfor incorporation into the primer comprise an acceptor fluorophore thatis activated by the donor when the two are in proximity.

Measured signals can be analyzed manually or preferably by appropriatecomputer methods to tabulate results. Preferably, the signals ofmillions of analogs are read in parallel and then deconvoluted toascertain a sequence. The substrates and reaction conditions can includeappropriate controls for verifying the integrity of hybridization andextension conditions, and for providing standard curves forquantification, if desired. For example, a control nucleic acid can beadded to the sample. The absence of the expected extension product is anindication that there is a defect with the sample or assay componentsrequiring correction.

As another example, the described nucleotide analogs can be used tofacilitate “four color” sequencing by synthesis if each base (A, C, G,T) is labeled with a dye emitting and/or absorbing at a different andresolvable wavelength. The sequencing procedure can be shortened fromfour separate addition cycles (i.e., one for each base) to thefollowing: add A, C, G, T (each differently labeled) with polymerase andan appropriate reaction buffer, rinse, image the four resolvable dyesand record which base (if any) was incorporated, cleave and cap thenucleotides, and repeat. The described nucleotide analogs facilitatethis kind of sequencing because of their ability to incorporate one andonly one base at a time. Without that ability, if all four bases areadded to the incorporation reaction at once multiple bases would beadded to a given strand and the interactions between the proximate dyeswould hinder the ability to resolve the sequence information correctly.

For example, the nucleotide analogs described herein can facilitatesequencing nucleic acids containing homopolymer sequences, usingsequencing by synthesis methodology (e.g., using the methods of US2007/0190546, herein incorporated by reference in its entirety for allpurpose. When the template sequence contains a homopolymer, using apolymerase, nucleotide analog, and reaction buffer combination thatallows for only a single nucleotide analog incorporation allows for eachbase in the homopolymer to be sequenced sequentially. After one base isincorporated into the homopolymer and detected, the portion of theanalog that inhibits subsequent base incorporation and that contains thefluorescent label is removed, making incorporation of the next base inthe homopolymer possible during the next addition cycle of the correctbase.

Reference to the following figures or schemes illustrating an exemplaryreaction scheme and nucleotide analogs is intended in no way to limitthe scope of this invention but is provided to illustrate how to prepareand use the compounds of the present invention.

EXAMPLES Example 1 Caproic-Glu and Caproic-Glu

3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionicacid 2,5-dioxo-pyrrolidin-1-yl ester (2)

To a solution of Fmoc-Cys(SStBu)-OH (1, 2.15 g, 5.0 mmole) dissolved inanhydrous CH₂Cl₂ (30 mL) was addedN-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC,1.146 g, 6 mmole), the reaction mixture was stirred for 10 min. at roomtemperature (RT) and then added N-hydroxysuccinimide (NHS) (0.690 g, 6.0mmole). To this reaction mixture was added catalytic amount ofN,N′-dimethlyaminopyridine and stirred at RT until completion ofreaction tested with TLC. The solvent was evaporated and the residueobtained was extracted with ethyl acetate (50 mL×2), washed with 1MNaHCO₃ (10 mL), followed by brine solution (20 mL) and dried overanhydrous Na₂SO₄. Evaporation of the solvent afforded 2 as a whitecrystalline solid. Yield. 2.5 g (95%).

6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-hexanoicacid (3)

To a solution of 6-Aminohexanoic acid (0.158.g, 1.2 mmole) dissolved in0.1M NaHCO₃ (2.0 mL) was added the NHS ester 2 (0.68 g, 1.3 mmole) in 4mL of anhydrous THF. The reaction mixture was stirred at RT for 2 hr.The solvent was completely evaporated and the dried solid residueobtained was dissolved in CH₃OH/CH₂Cl₂ mixture and purified by silicagel column chromatography using 10% CH₃OH/CH₂Cl₂ and obtained 3 as awhite solid on evaporation the solvent. Yield: 0.5 g (77%).

6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-hexanoicacid 2,5-dioxo-pyrrolidin-1-yl ester (4)

To a solution of (3, 500 mg, 0.92 mmole) dissolved in anhydrousCH₂Cl₂/THF (1:1) (5 mL) was addedN-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC, 191mg, 1.0 mmole), followed by NHS (115 mg, 1.0 mmole). To this reactionmixture was added catalytic amount of N,N′-dimethlyaminopyridine andstirred at RT until completion of reaction tested with TLC. The solventwas evaporated and the residue obtained was extracted with ethyl acetate(50 mLx2), washed with 1M NaHCO₃ (10 mL), followed by brine solution (10mL) and dried over anhydrous Na₂SO₄. Evaporation of the solvent afforded4 as a white crystalline solid. Yield. 0.52 g (88%).

2-{6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-hexanoylamino}-pentanedioicacid (5)

To a stirred solution of Glutamic acid (20 mg, 0.14 mmole) in 0.2MNaHCO₃ (0.5 mL) was added6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propionylamino]-hexanoicacid 2,5-dioxo-pyrrolidin-1-yl ester (4, 96 mg, 0.15 mmole) dissolved in(THF-DMF(1:1), 0.5 mL). The reaction mixture was stirred at RT for 10min. and analyzed with LCMS which showed the product (5) peak with massm/z: 671.95 [M-H]. The reaction was stirred at RT for overnight andpurified by HPLC using Phenomenex C18 preparative column, (250×21.00 mm,gradient: 2% CH₃CN/50 mM TEAB (triethylammonium bicarbonate), pH 8.4, 10mL/min flow). Fractions containing the compound 5 were collectedtogether and evaporated the solvent using rotary evaporator and dried.Yielded 5 as a white solid: 50 mg.

2-{6-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-mercapto-propionylamino]-hexanoylamino}-pentanedioicacid (6)

A solution of (5) (10 mg, 0.015 mmole) in H₂O-THF (1:1, 1.0 ml) wastreated with tris(2-carboxyethyl)phosphine (TCEP, 0.10 mL, 0.5M in H₂O).The reaction was stirred at RT for 4 h until complete cleavage ofdisulphide bond (monitored by LCMS) and purified by HPLC usingPhenomenex C18 preparative column, (250×21.00 mm, gradient: 2% CH₃CN/50mM TEAB, pH 8.4, 10 mL/min flow). Fractions containing the compound 6were pooled and used immediately for the subsequent displacementreaction with dATP-SPDP (SPDP: N-succinimidyl 3-(2-pyridyl dithio)propionate) and dCTP-SPDP as described below. LCMS: m/s: 583.95[M-H].

The fractions containing compound 6 in 60% CH₃CN/50 mM TEAB buffer (4.0mg, in 4 mL) collected from HPLC were mixed with dATP-SPDP (3.6 μmole,ref. previous patent) in 4 ml of 30% CH₃N/50 mM TEAB buffer, pH 8.4 in around bottom flask and stirred for 2 h. The reaction solution wasconcentrated under reduced pressure, diluted with water and purifiedwith HPLC (Phenomenex C18 column, 250×21.0 mm, gradient: 1.5% CH₃CN/50mM TEAB buffer, 10 mL/min flow rate). Fractions containing the desiredwere pooled together and evaporated and dried. Yielded 7 (3.0 mg) as awhite solid. LCMS: m/z: 2121.80 [M-2H], 606.05 [M/2-2H].

The compound 7 (2.0 mg) obtained was dissolved in anhydrous DMF (0.6 mL)added 60 μl of piperidine. The reaction mixture was then stirred at RTfor an hour. The complete cleavage of FMOC group was monitored by LCMSand the reaction mixture was purified by HPLC (Phenomenex C18 column,250×21.0 mm, gradient: 1.5% CH₃CN/50 mM TEAB buffer, 10 mL/min flowrate). Fractions containing the desired were pooled together andevaporated and obtained 8 (1.0 μmole) as a colorless solid. LCMS: 990.95[M/2-2H].

To a solution of 8 (0.5 μmole) in 0.5 mL of 50 mM K₂HPO₄ was addedCy5-NHS (1 mg, 1.2 μmole) dissolved in 20 μL of anhydrous DMF andstirred at RT until the complete disappearance of starting material 8which was monitored by LCMS. Then the blue color reaction mixture waspurified HPLC (Phenomenex C18 column, 250×21.0 mm, gradient: 1.5%CH₃CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions containing thedesired were pooled together and lyophilized. Yielded 9a (0.36 μmole) asa blue solid. LCMS: 814.40 [M/2-2H].

Similarly a solution of 8 (0.5 μmole) in 0.5 mL of 50 mM K₂HPO₄ wasadded Atto 647N—NHS (2 mg, 2.5 μmole) dissolved in 40 μL of anhydrousDMF and stirred at RT until the complete disappearance of startingmaterial 8 which was monitored by LCMS. Then the blue color reactionmixture was purified HPLC (Phenomenex C18 column, 250×21.0 mm, gradient:2.0% CH₃CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions containingthe desired were pooled together and lyophilized. Yielded 9b (0.3 μmole)as a blue solid. LCMS: 1595.2 [M-2H], 797.0 [M/2-2H].

The fractions containing compound 6 in 60% CH₃CN/50 mM TEAB buffer (3.0mg, in 3 mL) collected from HPLC were mixed with dCTP-SPDP (3.0 μmole,ref. previous patent) in 3 ml of 30% CH₃N/50 mM TEAB buffer, pH 8.4 in around bottom flask and stirred for 2 hr. The reaction solution wasconcentrated under reduced pressure, diluted with water and purifiedwith HPLC (Phenomenex C18 column, 250×21.0 mm, gradient: 1.5% CH₃CN/50mM TEAB buffer, 10 mL/min flow rate). Fractions containing the desiredwere pooled together and evaporated and dried. Yielded 10 (3.0 mg) as awhite solid. LCMS: m/z: 1189.85 [M-2H], 594.8 [M/2-2H].

The compound 10 (2.0 mg) obtained was dissolved in anhydrous DMF (0.6mL) added 60 μl of piperidine. The reaction mixture was then stirred atRT for an hour. The complete cleavage of FMOC group was monitored byLCMS and the reaction mixture was purified by HPLC (Phenomenex C18column, 250×21.0 mm, gradient: 1.5% CH₃CN/50 mM TEAB buffer, 10 mL/minflow rate). Fractions containing the desired were pooled together andevaporated and obtained 11 (1.2 μmole) as a colorless solid. LCMS:967.90 [M/2-2H].

To a solution of 11 (0.6 μmole) in 0.5 mL of 50 mM K₂HPO₄ was addedCy5-NHS (1.5 mg, 1.6 μmole) dissolved in 30 μL of anhydrous DMF andstirred at RT until the complete disappearance of starting material 11which was monitored by LCMS. Then the blue color reaction mixture waspurified HPLC (Phenomenex C18 column, 250×21.0 mm, gradient: 1.5%CH₃CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions containing thedesired were pooled together and lyophilized. Yielded 12a (0.5 μmole) asa blue solid. LCMS: 814.40 [M/2-2H].

Similarly a solution of 11 (0.4 μmole) in 0.5 mL of 50 mM K₂HPO₄ wasadded Atto 647N—NHS (2 mg, 2.5 μmole) dissolved in 40 μL of anhydrousDMF and stirred at RT until the complete disappearance of startingmaterial 11 which was monitored by LCMS. Then the blue color reactionmixture was purified HPLC (Phenomenex C18 column, 250×21.0 mm, gradient:2.0% CH₃CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions containingthe desired were pooled together and lyophilized. Yielded 12b (0.35μmole) as a blue solid. LCMS: 1595.2 [M-2H], 797.0 [M/2-2H].

Example 2 Caproic-Asp-Asp

α-N-Fmoc-S-tert-butylthio-L-cysteine (1 g, 2.32 mmol) was dissolved inanhydrous acetonitrile and solution of dicyclohexylcarbodiimide (DCC)(573 mg, 2.78 mmol in CH₃CN) was added followed by solution of NHS (345mg, 3.01 mmol in CH₃CN). After 1 hr. dicyclohexylurea was spun down andactive ester used without purification in coupling with ε-amino-hexanoicacid (304 mg, 2.32 mmol) dissolved in 50% aq. DMF.N,N′-Diisopropylethylamine (DIPEA) was added to correct pH to 8.0. Uponcompletion reaction mixture was acidified to pH 3 and partitionedbetween water and dichloromethane (DCM). Organic layer was dried overanhydrous Na₂SO₄ and evaporated to give 1.33 g of crude material.Purification using flash chromatography in DCM/methanol gave 745 mg ofpure material (MW=544.75).

α-N-Fmoc-S-tert-butylthio-L-cyst-caproic acid (3, 77 mg, 141 μmols,CH₃CN) was converted to NHS active ester using DCC (35 mg, 169 μmols,CH₃CN) and NHS (21 mg, 183 μmols, ACN). After 1 hr. precipitate ofdicyclohexylurea was removed by centrifugation and ester used withoutfurther purification in coupling with H-Asp-Asp-OH peptide (12 mg, 48nmols) dissolved in 0.5M K₂HPO₄, pH of reaction mixture corrected to 7.5with DIPEA. Progress of reaction was monitored by TLC (disappearance ofester) and by LC-MS (formation of product). Upon completion product wasisolated by direct injection on preparative HPLC (C18 column, 3% CH₃CNgradient in 50 mM TEAB, pH 8.6). Isolated product was lyophilized togive white powder (MW=774.9)

To free the thiol α-N-Fmoc-S-tert-butylthio-L-cyst-caproic-Asp-Asp-OH(15) was treated with 100 mM DTT in 0.1M K₂HPO₄ during 1 hr. at RT.Reaction was monitored by LC-MS and upon completion injected directly onpreparative HPLC (C18 column). Purification using 2% CH₃CN gradient in50 mM TEAB, pH 8.6 yielded product (MW=686.7) which was used immediatelywithout evaporation in displacement reaction with SPDP modifiednucleotide triphosphates.

dATP-AP3 and dCTP-AP3 were prepared by a modified procedure of Hobbs andCocuzza: a) Pyrophosphate and tributylamine were added to the reactionmixture rather than vice versa; b) After pyrophosphate addition thereaction was quenched with 50 mM TEAB within 15 min.; c) DEAE-Sephadexchromatography was replaced by preparative HPLC.

SPDP modification of dATP-AP3 and dCTP-AP3 was accomplished usingstandard protocol: 2 μmols of dNTP-AP3 were dissolved in 250 μl of 0.1NNaHCO₃ and 1.2 equivalent (eqv.) of freshly prepared 50 mM stock of SPDPin anhydrous DMF was added. Progress of modification was monitored usingLC-MS. Product was isolated using preparative HPLC (C18 column) with 1%CH₃CN gradient in 50 mM TEAB, pH 8.6 gradient and used in displacementreaction with thiol without evaporation of HPLC solvents (MW=717.01 fordCTP-AP3-SPDP, MW=740.03 for dATP-AP3-SPDP).

Small aliquots of isolated thiol were added to freshly isolateddNTP-AP3-SPDP to obtain displacement product. Progress of reaction wasmonitored by LC-MS after every addition of thiol. Reaction was completedwhen all dNTP-AP3-SPDP was consumed at which point reaction mixture wasconcentrated and purified on preparative HPLC (C18 column) using 1%gradient of CH₃CN in 50 mM TEAB, pH 8.6. Isolated product waslyophilized to give white powder (MW=1293.06 for cytidine-analog andMW=1316.09 for adenosine-analog).

Removal of Fmoc-protecting group was accomplished using 20% piperidinein CH₃CN (20 min., RT). Subsequently solvents were removed and crudereaction mixture purified on preparative HPLC (C18 column) using 2%CH₃CN gradient. Product was dried down and OD measured in water at 290nm for cytidine analog (800 nmols, MW=1070.8) and 280 nm for adenosineanalog (640 nmols, MW=1093.8).

Dye modified final products were prepared using following standardconditions: peptide modified dNTPs were re-dissolved in 20 mM K₂HPO₄ anddye-NHS dissolved in anhydrous DMF (5 mg in 100 μl) was added usinginitially 1.2 eqv. up to 4 eqv. to reach complete consumption ofstarting material. Progress of modification was monitored using LC-MS.Product was isolated using preparative HPLC (C18 column) with 1% CH₃CNgradient and 50 mM TEAB, pH 8.6. Desired fractions were combined,organic solvent removed under reduced pressure and products subjected toCH₃OH repurification on C18 HPLC column (1% CH₃OH gradient). Finalfractions were quantitated at 650 nm using ε₆₅₀=250000 M⁻¹cm⁻¹ for Cy5dye and 150000 M⁻¹cm⁻¹ for Atto 647N dye.

Example 3 Caproic-Arg-Arg-Arg

Compound 32

Compound 31 (100 mg, 0.18 mmol) was dissolved in 0.8 ml DMF and added0.2 mL piperidine and then kept at RT for 30 min. DMF was removed andthe residue was purified with flash column using CH₂Cl₂: CH₃OH (2:1).The purified amine (35 mg) was dissolved in 1 mL DMF and used directlyfor the next step without characterization. 3.5 mg of the purified aminein 0.1 mL DMF (10.8 μmol) was added 60 μL DMF and 40 μL DIPEA and thenCy5 Mono NHS Ester (6.63 μmol) in 100 μL anhydrous DMF was added intothe solution. After 30 minutes, the reaction mixture was purified withHPLC (Waters Delta 600 pump and 2487 Dual λ Absorbance Detector,Phenomenex C18 preparative column, 250×21.00 mm 10 micron, gradient:100% A for 5 min, then 1% B/min, buffer A 0.05 M TEAB, buffer B CH₃CN,10 mL/min flow). Fractions containing the desired compound 32 werepooled and quantified; (3.0 μmol, 45%, ε₆₄₉=250000); ESI-MS (negativeion mode): m/z=959.20 (M-H).

Compound 33

The NHS ester of the acid 32 was prepared by dissolving the acid 32 (3.0μmol) in DMF (500.0 μL) andN,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate(SbTMU) (4.3 mg, 12 μmol) in 100 μL DMF was added to the acid solutionfollowed by the addition of DIPEA (80 μL). After stirring at RT for 1hr., the reaction mixture was used immediately for peptide couplingwithout any purification. The peptide Arg-Arg-Arg-OH (14.5 mg, 30 μmol)was dissolved in 160 μL 0.5M phosphate buffer, and added to the freshlyprepared NHS ester of the acid 32. The reaction mixture was stirred for30 minutes and then the crude reaction mixture was purified with HPLC(Waters Delta 600 pump and 2487 Dual λ Absorbance Detector, PhenomenexC18 preparative column, 250×21.00 mm 10 micron, gradient: 100% A for 5min, then 1% B/min, buffer A 0.05 M TEAB, buffer B MeCN, 10 mL/minflow). Fractions containing the desired compound 33 were pooled andquantified; (0.6 μmol, 20%, ε₆₄₉=250000); ESI-MS (negative ion mode):m/z=713.45 [(M-2H)/2].

Compound 34

A solution of compound 33 (0.6 μmol) in 3 ml H₂O was treated with TCEP(300 μL, 1M solution) in an aluminum foil covered flask. After 30minutes, the reaction mixture was purified with HPLC (Waters Delta 600pump and 2487 Dual λ Absorbance Detector, Phenomenex C18 preparativecolumn, 250×21.00 mm 10 micron, gradient: 100% A for 5 min, then 1%B/min, buffer A 0.05 M TEAB, buffer B CH₃CN, 10 mL/min flow). Fractionscontaining the desired thiol, analyzed with ESI-MS (negative ion mode):m/z=669.90 [(M-2H)/2], were pooled and immediately added dATP-SPDP (1μmol in 1 mL H₂O). After 15 minutes, LCMS analysis indicated that thecompletion of the reaction and the reaction mixture was then partiallyconcentrated under reduced pressure to remove CH₃CN, then purified withHPLC (Waters Delta 600 pump and 2487 Dual λ Absorbance Detector,Phenomenex C18 preparative column, 250×10.00 mm 10 micron, gradient:100% A for 5 min, then 1% B/min, buffer A 0.05M TEAB, buffer B CH₃CN, 5mL/min flow). Fractions containing the desired compound were pooled andconcentrated and then purified again with HPLC using CH₃OH and TEABbuffer. The fractions containing the desired compound 34 were pooled andlyophilized to yield compound 34 as a bright blue solid (0.37 μmol, 62%,ε₆₄₉=250000). ESI-MS (negative ion mode): m/z=983.75 [(M-2H)/2].

Example 4 Cap-Asp-Asp-Asp-Asp

Compound 45

Cy5 Mono NHS Ester (100.0 μL, 6.63 μmol) in anhydrous DMF was added to asolution of amine 44 (13.26 μmol, 2 equiv) in DMF (100 μL) and DIPEA(20.0 μL) in an aluminum foil covered flask. After 30 minutes, thedisappearance of the starting amine was determined by LCMS or HPLC. Thereaction was HPLC purified (Waters Delta 600 pump and 2487 Dual λAbsorbance Detector, Phenomenex C18 preparative column, 250×21.00 mm 10micron, gradient: 100% A for 5 min, then 1% B/min, buffer A 0.05 M TEAB,buffer B CH₃CN, 10 mL/min flow). Fractions containing the desiredproduct were pooled and quantified; (4.0 mol, 60.3%, ε₆₄₉=250000);ESI-MS (negative ion mode): m/z=959.20 (M-H).

Compound 46

The NHS ester of the acid 45 was prepared by dissolving the acid 45 (4.0μmol, 1 eqv.) in DMF (700.0 μL) and the SbTMU 5.93 mg, 16.5 μmol, in 200μL DMF, 4.0 eqv.) was added, to the acid solution followed by theaddition of DIPEA (103.0 μL). After stirring at RT for 1 hour, thereaction mixture was used immediately for peptide coupling without anypurification. The peptide (Asp-Asp-Asp-Asp) was dissolved in DMF:H₂O(400.0 μL, 1:1), basified using DIPEA (50.0 μL). To this peptidesolution was added freshly prepared NHS ester of the acid 45. Thereaction mixture was stirred for 30 minutes and it was then analyzed byLCMS. The crude reaction mixture was HPLC purified (Waters Delta 600pump and 2487 Dual λ Absorbance Detector, Phenomenex C18 preparativecolumn, 250×21.00 mm 10 micron, gradient: 100% A for 5 min., then 1%B/min, buffer A 0.05 M TEAB, buffer B CH₃CN, 10 mL/min flow). Fractionscontaining the desired were pooled and quantified; (3.0 μmol, 75.0%,ε₆₄₉=250000); ESI-MS (negative ion mode): m/z=709.20 (1/2M-H).

Compound 47

A solution of compound 46 (1.0 μmol) in H₂O was treated with TCEP (40.0μL, 19.92 μmol, 0.5 M in H₂O, 19.92 equiv) in an aluminum foil coveredflask. After 30 minutes, the reaction mixture was analyzed by LCMS andwas then HPLC purified (Waters Delta 600 pump and 2487 Dual λ AbsorbanceDetector, Phenomenex C18 preparative column, 250×21.00 mm 10 micron,gradient: 100% A for 5 min., then 1% B/min, buffer A 0.05 M TEAB, bufferB CH₃CN, 10 mL/min flow). Fractions containing the desired were pooledand used immediately for the subsequent displacement reaction withoutremoving the solvent. ESI-MS (negative ion mode): m/z=665.45 (1/2M-H).

Compound 48a

HPLC fractions containing the thiol 7 (0.34 μmol, 1 eqv.) were mixedwith HPLC fractions containing dCTP-SPDP (0.41 μmol, 1.25 eqv.) in analuminum foil covered flask. After 15 min. LCMS analysis indicated thatthe completion of the reaction and it was then partially concentratedunder reduced pressure to remove CH₃CN, then HPLC purified (Waters Delta600 pump and 2487 Dual λ Absorbance Detector, Phenomenex C18 preparativecolumn, 250×10.00 mm 10 micron, gradient: 100% A for 5 min, then 1%B/min, buffer A 0.05 M TEAB, buffer B CH₃CN, 5 mL/min flow). Fractionscontaining the desired were pooled and lyophilized to yield compound Ias a bright blue solid (0.17 μmol, 50%, ±649=250000). The desiredproduct was HPLC purified a second time under the same conditions, usingCH₃OH instead of CH₃CN for buffer B. Fractions containing the desiredwere pooled and stored at −80° C. without removing the solvent. ESI-MS(negative ion mode): m/z=968.35 (1/2M-H).

Compound 49a

HPLC fractions containing thiol 47 (0.5 μmol, 1 eqv.) were mixed withHPLC fractions containing dATP-SPDP (0.6 μmol, 1.2 eqv.) in an aluminumfoil covered flask. After 15 min. LCMS analysis indicated that thecompletion of the reaction and it was then partially concentrated underreduced pressure to remove CH₃CN, then HPLC purified (Waters Delta 600pump and 2487 Dual λ Absorbance Detector, Phenomenex C18 preparativecolumn, 250×10.00 mm 10 micron, gradient: 100% A for 5 min., then 1%B/min, buffer A 0.05M TEAB, buffer B CH₃CN, 5 mL/min flow). Fractionscontaining the desired were pooled and lyophilized to yield compound 49aas a bright blue solid (0.35 μmol, 70%, ε₆₄₉=250000). The desired wasHPLC purified a second time under the same conditions, using CH₃OHinstead of CH₃CN for buffer B. Fractions containing the desired werepooled and stored at −80° C. without removing the solvent. ESI-MS(negative ion mode): m/z=980.10 (1/2M-H).

Example 5 Caproic-Asp

NHS Ester

Fmoc-Cys(StBu)-OH (2.0 g, 4.63 mmol, 1 eqv.) was dissolved in CH₃CN (10mL). DCC (1.2 g, 5.81 mmol, 1.26 eqv.) was added, followed by NHS (0.70g, 6.08 mmol, 1.31 eqv.) and the reaction was stirred at RT for 1 hr.White precipitate (DCU) began forming within five min. The reactionmixture was transferred to Eppendorf tubes and centrifuged to remove thewhite precipitate. The supernatant was then used in subsequent reactionswithout further purification.

Acid

6-Aminohexanoic acid (0.60 g, 4.57 mmol, 1 eqv.) was dissolved in1:1H₂O:DMF (6 mL total). DIPEA (0.016 mL) was added to keep the pH about8. NHS ester (4.63 mmol in 10 mL CH₃CN, 1.01 eqv.) was added to thereaction mixture in 1 mL aliquots over about 10 min. DIPEA (0.02 mL) wasadded after each aliquot to keep the reaction basic. After the firstaliquot of NHS ester was added, the reaction became cloudy, and additionof extra H₂O (0.2 mL) was needed to clear up the solution. The reactionwas stirred at RT for two hours, then quenched with 20 mL 10% HCl (aq.).The aqueous phase was extracted with CH₂Cl₂ (2×50 mL). The organic phasewas dried over Na₂SO₄, filtered, and concentrated under reduced pressureto yield a brown oil. Purification by flash column chromatography (100%CH₂Cl₂ to 5% CH₃OH/CH₂Cl₂) afforded the desired acid as a white foam(2.14 g, 86%).

NHS Ester

The starting acid (0.99 g, 1.82 mmol, 1 eqv.) was dissolved in CH₃CN (10mL). DCC (0.46 g, 2.23 mmol, 1.23 eqv.) was added, followed by NHS (0.28g, 2.43 mmol, 1.34 eqv.) and the reaction was stirred at RT for an hour.White precipitate (DCU) began forming within 5 min. The reaction mixturewas transferred to Eppendorf tubes and centrifuged to remove the whiteprecipitate. The supernatant was then used in subsequent reactionswithout further purification.

Dimethyl Ester

L-Aspartic acid dimethyl ester hydrochloride (0.2 g, 1.01 mmol, 2 eqv.)was dissolved in CH₃CN (1 mL) and DIPEA (0.32 mL, 1.84 mmol, 4 eqv.). Asolution of NHS ester (0.48 mmol, 1 eqv.) in CH₃CN (2 mL) was added, andthe reaction was stirred at RT for 12 hr. The reaction was diluted withEtOAc (25 mL), then washed with brine (1×30 mL) and sat. NH₄Cl (aq.)(1×30 mL). The organic phase was dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. Purification by flash columnchromatography (100% CH₂Cl₂ to 2% CH₃OH/CH₂Cl₂) afforded the desiredester as a white foam (0.12 g, 36%).

Diacid

1M LiOH(aq) (0.18 mL, 6 equiv) was added to a solution of dimethyl ester(0.02 g, 0.029 mmol, 1 eqv.) in THF (0.30 mL). The reaction was stirredat RT until the starting dimethyl ester was consumed based on LCMSanalysis (about 15 min). The crude reaction was then HPLC purified(Waters Delta 600 pump and 2487 Dual λ Absorbance Detector, PhenomenexC18 preparative column, 250×21.2 mm 10 micron, gradient: 90% A for 3min., then 5% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 10 mL/min.flow). Fractions containing the desired were pooled and concentrated toyield the desired diacid, which was used for subsequent reactionswithout quantifying.

Thiol

Diacid (˜29 μmol, 1 eqv.) was treated with TCEP (1.7 mL, 0.85 mmol, 0.5Min H₂O, 29 eqv.). The reaction was stirred at RT until the startingmaterial was consumed based on LCMS analysis (about 30 min.). The crudereaction was then HPLC purified (Waters Delta 600 pump and 2487 Dual λAbsorbance Detector, Phenomenex C18 preparative column, 250×21.2 mm 10micron, gradient: 100% A for 3 min, then 5% B/min., buffer A 0.05M TEAB,buffer B CH₃CN, 10 mL/min. flow). Fractions containing the desired werepooled and used for subsequent reactions without concentrating orquantifying.

Disulfide

HPLC fractions containing the thiol (about 10 μmol, 2 eqv.) were mixedwith HPLC fractions containing SPDP-dATP (5 μmol, 1 equiv). After theSPDP-dATP was consumed based on LCMS analysis (about 10 min), thereaction was partially concentrated under reduced pressure to removeCH₃CN and then HPLC purified (Waters Delta 600 pump and 2487 Dual λAbsorbance Detector, Phenomenex C18 preparative column, 250×15.0 mm 10micron, gradient: 100% A for 3 min, then 1% B/min., buffer A 0.05M TEAB,buffer B CH₃CN, 10 mL/min. flow). Fractions containing the desired werepooled and lyophilized, then used for subsequent reactions withoutquantifying.

Amine

The starting carbamate (˜5 μmol, 1 eqv.) was treated with 20% piperidinein 1:1 DMF: CH₃CN (2 mL), and stirred at RT until the starting materialwas consumed based on LCMS analysis (˜15 min). After removing thesolvent under reduced pressure, the reaction was HPLC purified (WatersDelta 600 pump and 2487 Dual λ Absorbance Detector, Phenomenex C18preparative column, 250×21.2 mm 10 micron, gradient: 100% A for 3 min,then 1% B/min., buffer A 0.05M TEAB, buffer B CH₃OH, 10 mL/min. flow).Fractions containing the desired were pooled and lyophilized to yieldthe product as a white foam (1 μmol, 20%, ε₂₈₀=12700).

A* Caproic-Asp

Atto647N—NHS ester (0.030 mL, 1.8 μmol, 0.06M in anhydrous DMF, 3.6eqv.) was added to a solution of amine (0.5 μmol, 1 eqv.) in H₂O (0.25mL) in 10 μL aliquots. The reaction was monitored by LCMS to determinehow much dye was needed to consume the starting amine. Afterdisappearance of amine, the crude reaction was HPLC purified (WatersDelta 600 pump and 2487 Dual λ Absorbance Detector, Phenomenex C18preparative column, 250×10.0 mm 10 micron, gradient: 100% A for 3 min,then 2% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 5 mL/min. flow).Fractions containing the desired were pooled and concentrated, then HPLCpurified a second time under the same conditions, using CH₃OH instead ofCH₃CN for buffer B. Fractions containing the desired were pooled andstored at −80° C. without removing the solvent (0.086 μmol, 17%,±645=150000).

Disulfide

HPLC fractions containing the thiol (˜10 mmol, 6 eqv.) were mixed withHPLC fractions containing SPDP-dGTP (1.5 μmol, 1 eqv.). After theSPDP-dGTP was consumed based on LCMS analysis (about 10 min), thereaction was partially concentrated under reduced pressure to removeCH₃CN and then HPLC purified (Waters Delta 600 pump and 2487 Dual λAbsorbance Detector, Phenomenex C18 preparative column, 250×10.0 mm 10micron, gradient: 100% A for 3 min., then 1% B/min, buffer A 0.05M TEAB,buffer B CH₃CN, 5 mL/min. flow). Fractions containing the desired werepooled and lyophilized, then used for subsequent reactions withoutquantifying.

Amine

The starting carbamate (˜1.5 μmol, 1 eqv.) was treated with 20%piperidine in DMF (0.5 mL), and stirred at RT until the startingmaterial was consumed based on LCMS analysis (about 15 min). Afterremoving the solvent under reduced pressure, the reaction was HPLCpurified (Waters Delta 600 pump and 2487 Dual λ Absorbance Detector,Phenomenex C18 preparative column, 250×10.0 mm 10 micron, gradient: 100%A for 3 min, then 1% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 5mL/min. flow). Fractions containing the desired were pooled andlyophilized to yield the product as a white foam (0.26 μmol, 17%,ε₂₇₂=11900).

G* Caproic-Asp

Atto647N—NHS ester (0.011 mL, 0.66 μmol, 0.06 M in anhydrous DMF, 2.5eqv.) was added to a solution of amine (0.26 μmol, 1 equiv) in H₂O (0.50mL) in small aliquots. The reaction was monitored by LCMS to determinehow much dye was needed to consume the starting amine. Afterdisappearance of amine, the crude reaction was HPLC purified (WatersDelta 600 pump and 2487 Dual λ Absorbance Detector, Phenomenex C18preparative column, 250×10.0 mm 10 micron, gradient: 100% A for 3 min,then 2% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 5 mL/min. flow).Fractions containing the desired were pooled and concentrated, then HPLCpurified a second time under the same conditions, using CH₃OH instead ofCH₃CN for buffer B. Fractions containing the desired were pooled andstored at −80° C. without removing the solvent (0.076 μmol, 29%,8645=150000).

Disulfide

HPLC fractions containing the thiol (about 5 μmol, 5 eqv.) were mixedwith SPDP-dCTP (1 μmol, 1 eqv.) in H₂O (0.20 mL). After the SPDP-dCTPwas consumed based on LCMS analysis (about 10 min.), the reaction waspartially concentrated under reduced pressure to remove CH₃CN and thenHPLC purified (Waters Delta 600 pump and 2487 Dual λ AbsorbanceDetector, Phenomenex C18 preparative column, 250×21.2 mm 10 micron,gradient: 100% A for 3 min, then 3% B/min., buffer A 0.05M TEAB, bufferB CH₃CN, 5 mL/min. flow). Fractions containing the desired were pooledand lyophilized, then used for subsequent reactions without quantifying.

Amine

The starting carbamate (about 1 μmol, 1 eqv.) was treated with 20%piperidine in CH₃CN (0.5 mL), and stirred at RT until the startingmaterial was consumed based on LCMS analysis (˜15 min). After removingthe solvent under reduced pressure, the reaction was HPLC purified(Waters Delta 600 pump and 2487 Dual λ % Absorbance Detector, PhenomenexC18 preparative column, 250×10.0 mm 10 micron, gradient: 100% A for 3min., then 1% B/min, buffer A 0.05M TEAB, buffer B CH₃CN, 5 mL/min.flow). Fractions containing the desired were pooled and lyophilized toyield the product as a white foam (0.15 μmol, 15%, ε₂₉₄=9300).

C* Caproic-Asp

Atto647N—NHS ester (0.012 mL, 0.72 μmol, 0.06M in anhydrous DMF, 3.6eqv.) was added to a solution of amine (0.15 μmol, 1 eqv.) in H₂O (0.20mL) in 5 μL aliquots. The reaction was monitored by LCMS to determinehow much dye was needed to consume the starting amine. Afterdisappearance of amine, the crude reaction was HPLC purified (WatersDelta 600 pump and 2487 Dual λ Absorbance Detector, Phenomenex C18preparative column, 250×10.0 mm 10 micron, gradient: 100% A for 3 min.,then 2% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 5 mL/min. flow).Fractions containing the desired were pooled and concentrated, then HPLCpurified a second time under the same conditions, using CH₃OH instead ofCH₃CN for buffer B. Fractions containing the desired were pooled andstored at −80° C. without removing the solvent (0.030 μmol, 20%,ε₆₄₅=150000).

Disulfide

HPLC fractions containing the thiol (˜5 μmol, 2.5 equiv) were mixed withSPDP-dUTP (2 μmol, 1 eqv.) in H₂O (0.13 mL). After the SPDP-dUTP wasconsumed based on LCMS analysis (˜10 min), the reaction was partiallyconcentrated under reduced pressure to remove CH₃CN and then HPLCpurified (Waters Delta 600 pump and 2487 Dual λ Absorbance Detector,Phenomenex C18 preparative column, 250×10.0 mm 10 micron, gradient: 100%A for 3 min., then 1% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 5mL/min. flow). Fractions containing the desired were pooled andlyophilized, then used for subsequent reactions without quantifying.

Amine

The starting carbamate (˜1 μmol, 1 equiv) was treated with 20%piperidine in DMF (2 mL), and stirred at RT until the starting materialwas consumed based on LCMS analysis (about 15 min). After removing thesolvent under reduced pressure, the reaction was HPLC purified (WatersDelta 600 pump and 2487 Dual λ Absorbance Detector, Phenomenex C18preparative column, 250×10.0 mm 10 micron, gradient: 100% A for 3 min,then 1% B/min., buffer A 0.05M TEAB, buffer B CH₃CN, 5 mL/min. flow).Fractions containing the desired were pooled and lyophilized to yieldthe product as a white foam (0.19 μmol, 19%, ε₂₈₉=13000).

T* Caproic-Asp

Atto647N—NHS ester (0.010 mL, 0.68 μmol, 0.06M in anhydrous DMF, 3.6eqv.) was added to a solution of amine (0.19 μmol, 1 eqv.) in H₂O (0.40mL) in small aliquots. 1M K₂HPO₄ (0.40 mL) was also added to acceleratethe reaction after there was little product formed within an hour. Thereaction was monitored by LCMS to determine how much dye was needed toconsume the starting amine. After disappearance of amine, the crudereaction was HPLC purified (Waters Delta 600 pump and 2487 Dual λAbsorbance Detector, Phenomenex C18 preparative column, 250×10.0 mm 10micron, gradient: 100% A for 3 min., then 2% B/min., buffer A 0.05MTEAB, buffer B CH₃CN, 5 mL/min. flow). Fractions containing the desiredwere pooled and concentrated, then HPLC purified a second time under thesame conditions, using CH₃OH instead of CH₃CN for buffer B. Fractionscontaining the desired were pooled and stored at −80° C. withoutremoving the solvent (0.059 μmol, 31%, ε₆₄₅=150000).

Example 6 Caproic-Asp-Asp-Asp-Asp (Alternative Routes)

Example 7 G* Pro-Pro-Lys-Pro-Asp

The schemes above and variations thereof may be utilized for synthesesof derivatives and analogs of the exemplary nucleotide analogs shownabove, for example, those having additional amino groups at theInhibitor end and/or compounds of different linking groups.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. Contemplated equivalents of thenucleotide analogs disclosed here include compounds which otherwisecorrespond thereto, and which have the same general properties thereof,wherein one or more simple variations of substituents or components aremade which do not adversely affect the characteristics of the nucleotideanalogs of interest. In general, the components of the nucleotideanalogs disclosed herein may be prepared by the methods illustrated inthe general reaction schema as described herein or by modificationsthereof, using readily available starting materials, reagents, andconventional synthesis procedures.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

INCORPORATION BY REFERENCE

The entire disclosure of each of the publications and patent documentsreferred to herein is incorporated by reference in its entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1. A method for sequencing a nucleic acid, the method comprising thesteps of: exposing a nucleic acid duplex comprising a template portionand a primer portion to a nucleotide analog comprising an inhibitor thatis charged or capable of becoming charged, and a polymerase, underconditions that permit template-dependent incorporation of the analoginto the primer; detecting incorporation of the analog; removing orneutralizing the inhibitor; and repeating the exposing, detecting, andremoving steps at least once, thereby to determine the sequence of thetemplate.
 2. The method of claim 1, wherein the template portion and/orthe primer portion is directly or indirectly anchored to a support. 3.The method of claim 1, wherein the detecting step comprises detectingindividual analogs.
 4. The method of claim 1 further comprising the stepof removing unincorporated analog.
 5. The method of claim 1, wherein theinhibitor is selected from the group consisting of one or morecarboxylic acid, one or more phosphate, one or more amino acid, one ormore peptide, one or more sulfate, one or more caproic acid, and anycombination thereof.
 6. The method of claim 5, wherein the amino acid isa negatively charged amino acid.
 7. The method of claim 6, wherein theamino acid is selected from the group consisting of aspartic acid,glutamic acid, histidine, lysine, and arginine.
 8. The method of claim5, wherein the peptide is from about 2 to about 10 amino acids inlength.
 9. The method of claim 1, wherein the inhibitor comprisesmultiple charged groups.
 10. The method of claim 1, wherein theinhibitor is negatively charged.
 11. The method of claim 1, wherein theinhibitor is positively charged.
 12. The method of claim 1, wherein theinhibitor does not cause steric inhibition of the polymerase.
 13. Anucleotide analog, comprising a nucleoside triphosphate; an inhibitorcomprising (a) one or more multiply charged groups or groups capable ofbecoming multiply charged, or (b) two or more singly charged groups ortwo or more groups capable of becoming singly charged; a detectablelabel; and a linker connecting the inhibitor and the label to thenucleoside triphosphate.
 14. The analog of claim 13, wherein the linkeris cleavable.
 15. The analog of claim 14, wherein after the linker iscleaved, the residual analog has the structure of:

wherein B¹ is selected from the group consisting of purine bases,pyrimidine bases, and derivatives of purine and pyrimidine bases; R′ isindependently selected from the group consisting of —OH, —O—P(O)(OH)₂,—O—C(O)—R^(x), —NHR^(y), and an —O-blocking agent, wherein R^(x) andR^(y) are alkyl groups; R″ is independently selected from the groupconsisting of H and —OH; R⁷ is a phosphodiester or a phosphoryl group;and z is an integer from about 1 to about
 5. 16. The analog of claim 13,wherein the charged groups consist of between about 2 to about 10charged groups.
 17. The analog of claim 13, wherein the charged groupsare selected from any combination of one or more carboxylic acid, one ormore phosphate, one or more amino acid, one or more peptide, one or moresulfate, and one or more caproic acid.
 18. The analog of claim 13,wherein the label is optically detectable.
 19. The analog of claim 18,wherein the label is a fluorescent label.
 20. The analog of claim 13,wherein the inhibitor is not a steric inhibitor of a polymerase enzyme.21. The analog of claim 13, wherein the nucleoside triphosphate isselected from ATP, GTP, CTP, TTP, UTP, dATP, dGTP, dCTP, dTTP, dUTP, oran analog of any of the foregoing.
 22. A nucleotide analog of thefollowing Formula II:

wherein NTP is a nucleoside or nucleotide triphosphate or an analog ofeither capable of incorporating onto the 3′ end of a polynucleotidestrand hybridized to a template presenting the complement of the NTP; Lis a detectable label that facilitates the identification of thenucleotide analog; Inhibitor comprises (a) one or more multiply chargedgroups or groups capable of becoming multiply charged, or (b) two ormore singly charged groups or two or more groups capable of becomingsingly charged; R₁ and R₂ are independently a bond or a group, whereinat least one of R₁ and R₂ comprises a cleavable bond, which uponcleavage results in de-association of NTP from both L and Inhibitor; R₃is a bond or group linking R₂ to the Inhibitor moiety; and R₄ is a bondor group linking R₂ to a L.
 23. The nucleotide analog of claim 22,wherein the Inhibitor does not comprise a nucleotide or nucleoside oranalogs thereof.
 24. The nucleotide analog of claim 22, wherein theInhibitor comprises a negatively charged group or a group capable ofbecoming negatively charged.
 25. The nucleotide analog of claim 22,wherein the Inhibitor comprises a positively charged group or a groupcapable of becoming positively charged.
 26. The nucleotide analog ofclaim 22, wherein the Inhibitor comprises two or more charged groups.27. The nucleotide analog of claim 22, wherein the Inhibitor comprises acharged group selected from the group consisting of —COOH, —PO₄, —SO₄,—SO₃, —SO₂, —NR_(w)R_(v), where R_(w) and R_(v) independently is H, analkyl or aryl group.
 28. The nucleotide analog of claim 22, wherein theInhibitor comprises

wherein R₈ and R₉ independently is a H or an alkyl group; each of x andy is an integer from 0 to about
 10. 29. The nucleotide analog of claim28, wherein R₈ and R₉ are H atoms and x=1 and y=2.
 30. The nucleotideanalog of claim 22, wherein the Inhibitor does not comprise a —PO₄group.
 31. The nucleotide analog of claim 22, wherein the Inhibitor doesnot comprise an aryl group.
 32. The nucleotide analog of claim 22,wherein the Inhibitor comprises an amino acid group or an amino acidanalog group.
 33. The nucleotide analog of claim 32, wherein theInhibitor comprises a peptide of 2 to 20 units of amino acids oranalogs.
 34. The nucleotide analog of claim 32, wherein the Inhibitorcomprises a group selected from the group consisting of Glu, Asp, Arg,His, Thr, Trp, Gln, Tyr and Lys.
 35. The nucleotide analog of claim 22,wherein R₃ comprises

wherein R₅ is a H or an alkyl group; p is an integer from 0 to about 10.36. The nucleotide analog of claim 35, wherein p is 5 or
 6. 37. Thenucleotide analog of claim 22, wherein R₁ comprises a C—C triple bond.38. The nucleotide analog of claim 22, wherein R₁ comprises a S—S bond.39. The nucleotide analog of claim 22, wherein R₁ comprises a C—C triplebond and a S—S bond.
 40. The nucleotide analog of claim 22, wherein R₁comprises

wherein R₆ is a H or an alkyl group; q and r independently is an integerfrom about 1 to about
 10. 41. The nucleotide analog of claim 40, whereinq is 1 or 2 and r is 1, 2 or
 3. 42. The nucleotide analog of claim 22,wherein NTP is selected from dATP, dGTP, dCTP, dTTP, dUTP, ATP, GTP,CTP, TTP, UTP or an analog thereof.
 43. The nucleotide analog of claim22, wherein the L is an optically-detectable moiety.
 44. The nucleotideanalog of claim 43, wherein the optically-detectable moiety comprises afluorophore.
 45. The nucleotide analog of claim 44, wherein thefluorophore is Cy5 or ATTO 647N.
 46. The nucleotide analog of claim 22,wherein the group of the Inhibitor that is charged or capable ofbecoming charged is from about 5 to about 60 bonds away from the NTP.47. The nucleotide analog of claim 36, wherein p is 5 and the detectablelabel comprises ATTO 647N.
 48. The nucleotide analog of claim 22,wherein R₃ comprises

wherein k is an integer from about 1 to about
 5. 49. The nucleotideanalog of claim 48, wherein the Inhibitor comprises a —COOH group. 50.The nucleotide analog of claim 49, wherein the Inhibitor comprises twoor more —COOH groups.
 51. The nucleotide analog of claim 22, wherein R₃comprises

wherein R¹, R² are independently H or alkyl groups, and may togetherform 3, 4, 5, or 6-member rings, and j is an integer from about 1 toabout
 5. 52. The nucleotide analog of claim 22, wherein R₁ comprises

wherein R¹, R², R³, and R⁴ are independently H or alkyl groups, and twoor more of which may together form one or more 3, 4, 5, or 6-memberrings, and j is an integer from about 1 to about 3.